1. Field of the Invention
The present invention relates to mass spectrometry and more particularly to the laser desorption of very large organic molecules using a time of flight (TOF) mass spectrometer.
2. Description of the Related Art
Mass spectrometry is an analytical technique for the accurate determination of molecular weights, the identification of chemical structures, the determination of the composition of mixtures and quantitative elemental analysis. For example, it is possible to accurately determine the molecular weights of organic molecules. It is also possible to determine the structure of the organic molecules based on the fragmentation pattern of the ion formed when the molecule is ionized. A quantitative elemental analysis of organic molecules and compounds requires obtaining precise mass values from a high resolution mass spectrometer.
One type of mass spectrometer obtains a mass spectrum by passing the ions (electrically charged atoms or molecules) through a magnetic field. The ions form a beam which, when they are of different masses, are deflected through different angles by the magnetic field. The magnetic field is varied (swept) and, at each field strength, ions pass through precision slits to be measured by an electrical detector (electrometer). However, primarily due to the limitations of magnetic field strength, it is impractical to measure molecules having a mass-to-charge ratio (m/Z) greater than about 15,000.
The organic molecules of greater mass which are non-volatile and thermally labile (decomposed by heat) are of great medical and commercial interest, as they include, for example, proteins, DNA, oligosaccharides, commercially important polymers and pharmaceuticals.
It has been suggested, in a series of articles published by "Hillenkamp-Karas", cited below, that large organic molecules, of about 10,000-100,000 Daltons, may be analyzed in a time of flight (TOF) mass spectrometer. Those articles describe that the molecules of interest are dissolved in an aqueous solution of nicotinic acid, in a ratio of one molecule of interest to 1000 nicotinic acid molecules. The solution is dried and placed on a sample probe tip that is inserted into a TOF mass spectrometer. The dried material on the tip is searched, using a microscope, for a suitable spot, and that spot is activated by a laser beam ("microprobe"). The laser beam wavelength is in the UV (ultraviolet) region (266 nm wavelength) and the beam size at the tip is 8 um diameter (Hillenkamp 1) or 10-50 um (Karas, 2,3). The molecules are desorbed and ionized by the laser beam and are formed into beams by a series of electrodes creating an electric field, typically of 1000 volts/cm. The ion beam is directed down a tube which is a vacuum chamber (spectrometer tube), generally having an equilibrium pressure in the order of 10.sup.-6 mm mercury. Ions of different masses require different times to transverse the spectrometer tube. The time the tip (target) is struck with a laser pulse is taken as time zero and the various times the ions arrive at the opposite end (the ion detector) are measured and displayed generally on a graph (the mass spectrum).
The frequency of the laser is chosen to match the absorption frequency of the solid matrix, principally of nicotinic acid, which exhibits strong absorption at 266 nm wave length. The laser pulses, of 15 ns pulse width and 266 nm wavelength, are obtained from a frequency quadrupled Q-switched ND-YAG solid crystal laser instrument.
The "Hillenkamp-Karas" articles are the following:
1. Hillenkamp, "Laser Desorption Mass Spectrometry: Mechanisms, Techniques and Applicatons"; Bordeaux Mass Spectrometry Conference Report, 1988, pages 354-362.
2. Karas and Hillenkamp, "Ultraviolet Laser Desorption of Proteins Up to 120,000 Daltons", Bordeaux Mass Spectrometry Conference Report, 1988, pages 416,417.
3. Karas and Hillenkamp, "Laser Desorption Ionization of Proteins With Molecular Masses Exceeding 10,000 Daltons", Analytical Chemistry, 60, 2299, July 1988.
4. Karas, Ingendoh, Bahr and Hillenkamp, "UV-Laser Desorption/Ionization Mass Spectrometry of Femtomol Amounts of Large Proteins", Biomed. Environ. Mass Spectrum. (in press)
Although the previously described Hillenkamp-Karas articles are a real advance in the field, there are a number of problems and limitations to the methods.
The resolution of the mass spectrum is not as sharp as is possible, at much lower molecular weights, with magnetic field mass spectrometry. The Hillenkamp-Karas graphs show what appear to be a broad envelope of mass weights rather than the sharp peaks, which are desired. The work so far published by Hillenkamp and Karas on nicotinic acid assisted UV laser desorption shows spectral peaks with resolutions of less than about 50 Full Width at Half-Maximum definition (FWHM).
In addition, the procedure is time-consuming and costly. One must obtain a suitable spot on the tip using a microscope, by trial and error, and a number of attempts may be made before a successful spot is found. The instruments required to be used (laser microprobes and LAMMA) are relatively costly and complex. They have only studied positive ions, although negative ions sometimes provide complementary and/or unique information.
The wavelength published by Karas-Hillenkamp, in some cases, presents problems as to some molecules because that wavelength causes undesirable fragmentation of the molecule. It is difficult to simply change the wavelength with the teaching of the Karas-Hillenkamp articles, because the matrix (nicotinic acid) will only effectively absorb laser energy in a restricted range of wavelengths (below about 300 nm).
The use of laser beams in time of flight mass spectrometers is shown, for example, in U.S. Pat. Nos. 4,694,167; 4,686,366 and 4,295,046, incorporated by reference herein.